The development of immunoassays and advances in nucleic acid detection have advanced the art of the detection of biological samples. The enzyme-linked immunosorbent assay (ELISA) allows for the high throughput screening of samples for the presence of proteins in samples. The presence of the analyte is frequently detected by the use of an enzymatic, colorimetric assay based on alkaline phosphatase or horseradish peroxidase. This limits the sensitivity and the range of the assay depending on the range of detection of colorimetric changes of the enzyme substrate. This requires either initial screening to determine an approximate amount of the analyte in the serum, or the use of a large series of dilutions to ensure that a sample is tested within the detection range of the specific methods and reagents used. Other problems with this method include high background levels as well as low sensitivity. For example, direct binding of horseradish peroxidase to an ELISA plate results in unspecific background signal. To reduce unspecific binding, blocking solutions containing relatively inert proteins (milk or serum albumin) are added to the assay. However, the unspecific binding resulting in a background signal is not eliminated, thereby resulting in lower sensitivity of the assay.
To address these limitations, nucleic acid based detection methods for use in conjunction with enzyme-based detection of analytes in samples have been developed. Several so called “immuno-PCR assays” are known in the art and combine aspects of an ELISA assay with PCR. The assays produce a detectable signal when the probes/antibodies bind an analyte in a sample allowing for the amplification of a target nucleic acid.
For example, U.S. Pat. No. 5,665,539 combines detection with an antibody and the polymerase chain reaction (PCR) to increase sensitivity for the detection of a specific protein. In a standard immuno-PCR protocol, an antibody attached to a nucleic acid sequence binds to an epitope on an antigen molecule. The attachment between the antibody and nucleic acid occurs via a linker with bispecific affinity for nucleic acids and antibodies, thus resulting in the formation of a specific antigen-antibody-DNA conjugate. Subsequently, a segment of the attached nucleic acid sequence is amplified by PCR and the PCR products are detected by gel electrophoresis. However, linking DNA to antibodies has been problematic because DNA is sticky and any unbound DNA is not easily washed from the system prior to detection, giving rise to non-specific binding and high background in the assay.
Other such assays are described in U.S. Publication No. 2002/0132233, U.S. Pat. No. 5,985,548, U.S. Publication No. 2005/0026161 and U.S. Publication No. 2005/0239108. U.S. Patent Publication Nos. 2002/0132233, 2005/0026161, and 2005/0239108 are directed to sandwich immuno-PCR methods using analyte-specific antibodies coupled directly or indirectly (e.g., using a biotin/avidin binding pair) to an oligonucleotide. Binding of the antibody to the analyte in the sample results in an analyte-antibody-DNA complex. Antibody binding is detected by subjecting the complex to amplification conditions, where the attached oligonucleotide is used as a template to generate an amplification product. None of these methods, however, couples an antibody, or other binding molecule, with a polymerase. Rather they all require the use of an antibody coupled directly or indirectly to an oligonucleotide. Non-specific binding of the antibody/DNA conjugate contributes to high background and interferes with the ability to detect small quantities of analyte. In addition, none of the methods uses two different polymerase reactions to generate the amplification product and increase the sensitivity of the assay.
U.S. Pat. No. 5,985,548 discloses another sandwich immuno PCR method in which a binding molecule, such as an antibody, is conjugated to a target oligonucleotide. As with the immuno-PCR methods discussed above, non-specific binding of the antibody/DNA conjugate contributes to high background and interferes with the ability to detect small quantities of analyte. In another embodiment, U.S. Pat. No. 5,985,548 discloses a sandwich immuno PCR method in which a binding molecule, such as an antibody, is conjugated to an enzyme, such as horseradish peroxidase, that can activate a moiety (e.g., tyramine) on the target oligonucleotide. Once the tyramine is activated, it forms a reactive intermediate that binds to a receptor on the solid support, thereby immobilizing the target oligonucleotide. The immobilized target oligonucleotide is then subjected to an amplification reaction. Although this embodiment avoids the use of reporter conjugates comprising DNA linked to an antibody or other binding moiety, it requires the use of a target nucleic acid having a moiety that is activated by the enzyme attached to the reporter conjugate and that upon activation forms a reactive intermediate that must bind to an immobilized receptor before amplification. Furthermore, none of the methods in U.S. Pat. No. 5,985,548 uses two different polymerase reactions to generate the amplification product.